双光子荧光显微镜作为一种高分辨光学仪器, 已经被广泛应用于生物样品的非侵入式三维光学成像中。 相比共聚焦显微镜, 双光子荧光显微镜拥有更深的探测深度。 然而, 即便如此, 在对较厚的生物样品进行非侵入式光学三维成像时, 样品的成像质量也往往会随着探测深度的增加而下降。 在临床和生物学领域对研究母性遗传起重要作用的小鼠卵母细胞拥有较大的直径(80～100 μm), 吸收和散射效应较为明显。 本文研究小鼠卵母细胞染色体的三维双光子荧光图像随探测深度增加图像质量的衰减程度。 通过对所得图像进行轴向衰减矫正, 利用体积作为参数, 将矫正前后小鼠卵母细胞内染色体三维双光子荧光图像进行对比。 结果表明, 由于吸收和散射效应, 卵母细胞存在较严重的光学轴向衰减问题, 因此, 对用双光子荧光三维成像手段获得的小鼠卵母细胞图像进行衰减矫正是有必要的。 这为进一步精确定量的研究卵母细胞内染色体的三维构像打下良好的基础。

As an optical microscope with high resolution, two-photon excitation (TPE) fluorescence microscope is widely used in noninvasive 3D optical imaging of biological samples. Compared with confocal laser scanning microscope, TPE fluorescence microscope provides a deeper detecting depth. In spite of that, the image quality of sample always declines as the detecting depth increases when a noninvasive 3D optical imaging of thicker samples is performed. Mouse oocytes with a large diameter, which play an important role in clinical and biological fields, have obvious absorption and scattering effects. In the present paper, we performed compensation for two-photon fluorescence images of mouse oocyte chromosomes. Using volume as a parameter, the attenuation degree of these chromosomes was also studied. The result of our data suggested that there exists a severe axial intensity loss in two-photon microscopic images of mouse oocytes due to the absorption and scattering effects. It is necessary to make compensation for these images of mouse oocyte chromosomes obtained from two-photon microscopic system. It will be specially needed in studying the quantitative three-dimensional information of mouse oocytes.